Topoisomerases are cellular enzymes that are crucial for replication and transcription of the cellular genome. Topoisomerases cleave the DNA backbone, thereby allowing topological change for replication and transcription of the cellular genome to occur, after which topoisomerases reseal the DNA backbone (Wang, Ann. Rev. Biochem. 65: 635 (1996)). Topoisomerases are efficient because DNA breakage is accompanied by covalent bonding between the enzyme and the DNA to create an intermediate that is resolved during the resealing step. This mechanism, while elegant, makes topoisomerases potentially dangerous. If the resealing step fails, a normally transient break in DNA becomes a long-lived disruption, one with a topoisomerase covalently joined to it. Unless a way is found to restore the continuity of the DNA the cell will die.
In virtually all topoisomerases, the heart of the covalent complex is a phosphodiester bond between a specific tyrosine residue of the enzyme and one end of the break (the 3′ end for eukaryotic topoisomerase I and the 5′ end for topoisomerases II and III). The high-energy nature of this bond normally ensures the resealing step.
Failure of resealing is dramatically increased by several drugs, such as camptothecin, a promising anti-cancer agent that specifically targets eukaryotic topoisomerase I (Chen et al., Ann. Rev. Pharmacol. Toxicol. 34: 191 (1994)). Protein-linked breaks also accumulate when topoisomerases act on DNA containing structural lesions like thymine dimers, abasic sites and mismatched base pairs (Pommier et al., Biochim. Biophys. Acta 1400: 83 (1998)). To the extent that such lesions arise during the normal life of a cell, topoisomerase-associated damage may be unavoidable.
Repair of topoisomerase-DNA covalent complexes is of obvious value to the cell but, until the present invention, very little was known about the mechanisms involved in such repair. Hydrolysis of the bond joining the topoisomerase to DNA had been proposed as a way to effect release of the topoisomerase such that the cleaved DNA could undergo conventional modes of break repair (Friedberg et al., DNA Repair and Mutagenesis (ASM Press, Washington, D.C. (1995)); Kanaar et al., Trends Cell. Biol. 8: 483 (1998)). Although no such hydrolysis has been reported for covalent complexes between DNA and topoisomerase II or III, such hydrolysis has been described for covalent complexes between DNA and topoisomerase I (Yang et al., PNAS USA 93: 11534 (1996)).
The present invention seeks to provide the enzyme responsible for hydrolysis of the covalent complexes between DNA and topoisomerase I, specifically tyrosine-DNA phosphodiesterase, which acts on a tyrosine linked through the side-chain oxygen to the 3′ phosphate of DNA. This and other objects and advantages, as well as additional inventive features, will become apparent to one of ordinary skill in the art from the detailed description provided herein.